昆明小鼠心室肌细胞分离方法及动作电位、L型钙通道电流记录

Isolation of Calcium-tolerant Cardiomyocytes from KM Mouse and Recording of Action Potential and the Currents of L-type Calcium Channels

关键词:  昆明小鼠  心肌细胞  膜片钳  动作电位  L型钙电流

Key words:  KM mouse  ventricular myocyte  patch clamp technique  action potentials  L-type calcium channel currents

摘要:

目的 探讨耐钙昆明小鼠心室肌细胞的急性分离方法及动作电位、L型钙通道电流的记录。方法 采用三步灌流法，首先灌流无钙台氏液，再换成含Ⅱ型胶原酶0.1 mg·mL^-1、胰蛋白酶0.01 mg·mL^-1、牛血清白蛋白0.2 mg·mL^-1的无钙台氏液灌流，消化液灌流期间，每隔5 min加入20 mL 的20 mmol·L^-1 CaCl₂，以观察流出液是否有单个心肌细胞来判断消化终点，最后灌流含1 mg·mL^-1牛血清白蛋白的KB液，采用全细胞膜片钳记录方式记录动作电位及L型钙通道电流。结果 获得80%~90%杆状心肌细胞，复钙后，仍有60%细胞保持静止，细胞表面干净整洁，折光性强，边缘和横纹清晰，立体感强，获得60%左右的耐钙心室肌细胞，并记录到典型的动作电位、L型钙通道电流。结论 该分离方法分离的细胞具有耐钙性和正常电生理特性。

Abstract:

OBJECTIVE To explore and establish simple and reliable method of isolating single calcium-tolerant ventricular myocytes of KM mouse for patch clamping and recording of action potential and the currents of L-type calcium channels. METHODS The three-step enzymatic dissociation method was used to isolate myocytes from ventricular tissue by the Langendorff apparatus. Hearts were perfused retrogradely with Ca²⁺ free Tyrode’s solution initially, and then with Ca2+ free Tyrode’s solution contained 0.1 mg·mL^-1 collagenase Ⅱ, 0.01 mg·mL^-1 trypsin and 0.2 mg·mL^-1 bovine serum albumin. During the perfusions, 20 mL 20 mmol·L^-1 of CaCl₂ was added to the digestive juice every 5 minutes. The terminal of digestion was judged by observing the existence of single myocyte in the efflux solution. KB solution contained 1 mg·mL^-1 bovine serum albumin was ultimately used. The action potential and the currents of L-type calcium channels were recorded by patch clamp in the entire cell mode. RESULTS The 80%-90% cells obtained were rod-shaped myocytes. After the recalcification, 60% cells stayed still, and the action potentials and L-type calcium channel currents could be successfully recorded. CONCLUSION The method is economical and effective, the myocytes obstaied in this way are suitable for the recording of patch clamp technique.